The Mosquito Larvicidal Activity of Lignans from Branches of Cinnamomum camphora chvar. Borneol

The chemical investigation of branches of Cinnamomum camphora chvar. Borneol guided by mosquito larvicidal activity led to the isolation of fourteen known lignans (1–14). Their structures were elucidated unambiguously based on comprehensive spectroscopic analysis and comparison with the literature data. This is the first report of these compounds being isolated from branches of Cinnamomum camphora chvar. Borneol. Compounds 3–5 and 8–14 were isolated from this plant for the first time. All compounds isolated were subjected to anti-inflammatory, mosquito larvicidal activity and cytotoxic activity evaluation. Compounds (1–14) showed significant mosquito larvicidal activity against Culex pipiens quinquefasciatus with lethal mortality in 50% (LC50), with values ranging from 0.009 to 0.24 μg/mL. Among them, furofuran lignans(1–8) exhibited potent mosquito larvicidal activity against Cx. p. quinquefasciatus, with LC50 values of 0.009–0.021 μg/mL. From the perspective of a structure–activity relationship, compounds with a dioxolane group showed high mosquito larvicidal activity and have potential to be developed into a mosquitocide.


Introduction
Mosquitoes transmit various diseases such as malaria, which caused 409,000 deaths in 2019 [1]. Culex pipiens quinquefasciatus is widely distributed south of Yangtze river in China and is known as the Japanese encephalitis virus (JEV) vector in China [2]. Because of the lack of vaccines, vector control has been considered as an effective approach to reducing mosquito-borne cases [3]. However, the extensive use of limited available chemicals has caused increasing resistance. For example, Cx. p. quinquefasciatus has become more or less resistant to permethrin, deltamethrin, temephos, chlorpyrifos, malathion and dieldrin in La Réunion Island [4].
Compounds 3-5 and 8-14 were first isolated from the titled plant and structures of all compounds were identified based on NMR spectroscopic methods, mass spectrometry, as well as by comparison with the literature data (See the Figures S1-S42 in Supplementary Information). Compounds 1-8 were furan lignans with the same basic mother nucleus and the relative configuration subtypes of compounds 2-6 were decided as trans- (H-7, 8, 8′, 9) by the shift difference between two protons at position 7'and the shift difference between two protons at position 9 (ΔδH-7′ and ΔδH-9 =0.3~0.4) [19]. More specifically, the structural differences of compounds 1-3 were reflected in the substitution of hydroxyl and methoxy groups on the benzene ring. Compounds 4-5 and 6-8 differed structurally from compounds 1-3 in that they contained one or two methylenedioxy groups. Different similar compounds with different substituents may have led to different biological activities.    Compounds 3-5 and 8-14 were first isolated from the titled plant and structures of all compounds were identified based on NMR spectroscopic methods, mass spectrometry, as well as by comparison with the literature data (See the Figures S1-S42 in Supplementary Information). Compounds 1-8 were furan lignans with the same basic mother nucleus and the relative configuration subtypes of compounds 2-6 were decided as trans-(H-7, 8, 8 , 9) by the shift difference between two protons at position 7 and the shift difference between two protons at position 9 (∆δ H-7 and ∆δ H-9 =0.3~0.4) [19]. More specifically, the structural differences of compounds 1-3 were reflected in the substitution of hydroxyl and methoxy groups on the benzene ring. Compounds 4-5 and 6-8 differed structurally from compounds 1-3 in that they contained one or two methylenedioxy groups. Different similar compounds with different substituents may have led to different biological activities.

Discussion
Compounds 1-9, 13 and 14 are furofuran lignans that feature with a bicyclic oxygen skeleton; these mainly showed antioxidant, insecticidal, inhibitory activity against AChE and NO production in LPS-treated BV-2 microglial cells [20]. Among them, Sesamin (7) has the effect of lowering cholesterol in clinical applications [20,21]. Two dibenzylbutane lignans (10)(11) were also isolated from Virola Venosa, but their biological activity has been

Discussion
Compounds 1-9, 13 and 14 are furofuran lignans that feature with a bicyclic oxygen skeleton; these mainly showed antioxidant, insecticidal, inhibitory activity against AChE and NO production in LPS-treated BV-2 microglial cells [20]. Among them, Sesamin (7) has the effect of lowering cholesterol in clinical applications [20,21]. Two dibenzylbutane lignans (10-11) were also isolated from Virola Venosa, but their biological activity has been
In a sense, as the main characteristic component of the branches of Cinamomum camphora chvar. Borneol, lignans may play a certain role in ecology such as protecting themselves from mosquitoes and pests. Among them, furofuran lignans(1-8) exhibited potent mosquito larvicidal activity against Cx. p. quinquefasciatus, with LC 50 values of 0.009-0.021 µg/mL; these values are far stronger than compounds dibenzylbutane lignans (10)(11) and benzodihydrofuran neolignan balanophonin (12), thus indicating the presence of a dioxolane group in compounds enhancing mosquito larvicidal activity. From the perspective of a structure-activity relationship, the mosquito larvicidal activity against Cx. p. quinquefasciatus in comparison to compounds 1-3, 4 and 6, 6-8 shows that there is no effect on the methoxy substitutions at the 3 and 3 sites of the benzene ring. There is also no effect on whether the 3 and 5 methoxy groups form a ring, but the hydroxyl substitution and the formation of double bonds at position 9 have a significant effect. It is necessary to conduct further research on the resistance of lignans to mosquitoes and insects, especially furan lignan analogues with a methylenedioxy group such as structural optimization. This will determine whether it is related to configuration or whether it is related to bioecology.

Biological Assays
Larvicidal bioassays were conducted based on the WHO requirement with slight modification [27]. Serial concentrations (10, 20, 40, 60, 80 and 100 mg/L) were tested for lignans. Thirty 4th instar larvae were tested in a 150 mL glass beaker with 100 mL of sterilized water and 5 replicates were conducted. Mortality was recorded after 24 h of treatment, and no food was provided during the treatment. Dimethyl sulfoxide (DMSO) was set as the negative control and permethrin was set as the positive control. The antitumor activity of tested compounds against HepG2 was performed by the MTT method [28]. The anti-inflammatory activity was evaluated by the inflammatory model of LPS-induced RAW264.7 macrophages [29].

Statistic Analysis
SPSS (version 19.0) was used to perform the statistical analyses. Standard probit analysis was conducted for the Cx. p. quinquefasciatus larvicidal bioassay and LC 50 values were calculated after 24 h of exposure. Significant differences in LC 50 values (p ≤ 0.05) were concluded only if there was no overlap in the confidence intervals.